C6 C1 Research Articles

Alkaloid biosynthesis. XVI. Colchicine: origin of the tropolone ring and studies with the C 6 -C 3 -C 6 -C 1 system.

Colchicine (1), isolated from Colchicum autumnale plants which had been fed with (±)-[3-14C]tyrosine, is degraded to prove that ca. 85% of its total activity is located in the tropolone ring at C-12. It is thereby established that the tropolone system is generated from the aromatic nucleus of tyrosine by a ring-expansion process with inclusion of the benzylic carbon atom. A biosynthetic scheme for colchicine based upon a C6–C3–C6–C1 precursor is outlined and is tested by experiments with labelled 1 -(5-hydroxy-2-hydroxymethyl-4-methoxyphenyl)-3-(3-hydroxy-4,5-dimethoxyphenyl)propylamine (10). This is not incorporated into colchicine by the plants and the implications of this finding are discussed. The lactone prepared by treatment of the Windaus anhydride (7-benzamido-8,9-dihydro-2,3,4-trimethoxy-7H-benzocycloheptene-5,6-dicarboxylic anhydride)(5) with hydriodic acid is 7-benz-amido-8,9-dihydro-2,3-dihydroxy-7H-benzocycloheptene-5,4-carbolactone (6).

Studies of enzyme-mediated reactions. I. Syntheses of deuterium- or tritium-labelled (3S)-and (3R)-phenylalanines: stereochemical course of the elimination catalysed by L-phenylalanine ammonia-lyase.

Two rational syntheses of L-phenylalanine [(2S)-isomer] have been devised which allow labelling with isotopic hydrogen at C-3 in configurations established by chemical correlation with [2-2H1]succinic acid. The labelled samples have been used to prove that L-phenylalanine ammonia-lyase eliminates the 3-pro-S hydrogen atom of L-phenylalanine together with ammonia to give trans-cinnamic acid (a) with the isolated enzyme from potatoes and (b) during the biosynthesis of colchicine in intact Colchicum autumnale plants.The biochemical formation of the C6–C1 unit required for the construction of haemanthamine (30) in Amaryllidaceae plants involves loss of the hydrogen atom from C-3 of cinnamic acid.

Relative stereochemistry of protonation and hydroxylation in the biosynthesis of lycorenine and haemanthidine from protocatechualdehyde

Tracer experiments show that in the biosynthesis of lycorenine and haemanthidine the hydrogen introduced in the incorporation of protocatechualdehyde into the aromatic C6–C1 unit of O-methylnorbelladine is the one removed in the hydroxylation.

Biosynthesis of phytoquinones. Biosynthetic origins of the nuclei and satellite methyl groups of plastoquinone, tocopherols and tocopherolquinones in maize shoots, bean shoots and ivy leaves.

1. By using dl-[ring-(14)C]phenylalanine, dl-[beta-(14)C]phenylalanine, dl-[alpha-(14)C]-tyrosine and dl-[beta-(14)C]tyrosine it was shown that in maize shoots (Zea mays) the nucleus and one nuclear methyl group of each of the following compounds, plastoquinone, gamma-tocopherol (aromatic nucleus) and alpha-tocopherolquinone, are formed from the nuclear carbon atoms and beta-carbon atom respectively of either exogenous phenylalanine or exogenous tyrosine. With ubiquinone only the aromatic ring of the amino acid is used in the synthesis of the quinone nucleus. Chemical degradation of plastoquinone and gamma-tocopherol molecules labelled from l-[U-(14)C]tyrosine established that a C(6)-C(1) unit directly derived from the amino acid is involved in the synthesis of these compounds. Radioactivity from [beta-(14)C]cinnamic acid is not incorporated into plastoquinone, tocopherols or tocopherolquinones, demonstrating that the C(6)-C(1) unit is not formed from any of the C(6)-C(1) phenolic acids associated with the metabolism of this compound. 2. The incorporation of radioactivity from l-[U-(14)C]tyrosine, dl-[beta-(14)C]tyrosine and dl-[U-(14)C]phenylalanine into bean shoots (Phaseolus vulgaris) and dl-[beta-(14)C]tyrosine and l-[Me-(14)C]methionine into ivy leaves (Hedera helix) was also investigated. Similar results were obtained to those reported for maize, except that in beans phenylalanine is only used for ubiquinone biosynthesis. This is attributed to the absence of phenylalanine hydroxylase from these tissues. In ivy leaves it is found that the beta-carbon atom of tyrosine gives rise to the 8-methyl group of delta-tocopherol, and it is suggested that for all other compounds examined it will give rise to the nuclear methyl group meta to the polyprenyl unit. 3. Preliminary investigations with the alga Euglena gracilis showed that in this organism ring-opening of tyrosine occurs to such an extent that the incorporation data from radiochemical experiments are meaningless. 4. The above results, coupled with previous observations, are interpreted as showing that in higher plants the nucleus of ubiquinone can be formed from either phenylalanine or tyrosine by a pathway involving as intermediates p-coumaric acid and p-hydroxybenzoic acid. Plastoquinone, tocopherols and alpha-tocopherolquinone are formed from p-hydroxyphenylpyruvate by a pathway in which the aromatic ring and C-3 of the side chain give rise respectively to the nucleus and to one nuclear methyl group. 5. Dilution experiments provided evidence that in maize shoots p-hydroxyphenylpyruvic acid and homogentisic acid (produced from p-hydroxyphenylpyruvic acid) are involved in plastoquinone biosynthesis, and presumably the biosynthesis of related compounds: however, other possible intermediates in the conversion including toluquinol (the aglycone of the proposed key intermediate) showed no dilution effects. Further, radioactivity from [Me-(14)C]toluquinol is not incorporated into any of the compounds examined. 6. Dilution experiments with 3,4-dihydroxybenzaldehyde and radioactive-labelling experiments with 3,4-dihydroxy[U-(14)C]benzoic acid demonstrated that these compounds are not involved in the biosynthesis of either ubiquinone or phylloquinone in maize shoots. 7. Evidence is also presented to show that in maize shoots ring-opening of the aromatic amino acids takes place. The suggestion is offered that this may take place via homogentisic acid, as in animals and some micro-organisms.

Constitution and biosynthesis of capsaicin

Capsaicin, the crystalline pungent principle of Capsicum annuum, has been shown to be a mixture of at least five closely related vanillyl amides. Phenylalanine, various hydroxylated cinnamic acids, and vanillylamine, all labelled with tritium in the aromatic ring, were incorporated biosynthetically into the C6–C1 unit of capsaicin. Tyrosine was incorporated much less efficiently than phenylalanine. The radioactive capsaicin mixture derived biosynthetically from phenylalanine was found to contain most of the activity in the minor, saturated components.